EP2 and EP3 receptors as therapeutic targets for underactive bladder/detrusor underactivity due to diabetic cystopathy in a type 1 diabetic rat model.

OBJECTIVES: Diabetic cystopathy (DC) is recognized as one of the major etiologies of underactive bladder (UAB)/detrusor underactivity (DU). Although DC was first reported about three decades ago, there is a distinct lack of effective pharmacological management methods for UAB/DU due to DC with a robust certainty of evidence. In this study, we investigated whether EP2 and EP3 receptors are promising targets of pharmacological management of UAB/DU due to DC. METHODS: We used streptozotocin (STZ)-induced diabetic Sprague-Dawley rats with postvoid residual urine (PVR) greater than 0.1 mL. Sixteen weeks after induction of diabetes, we performed awake single cystometry after oral administration of the vehicle, an α-blocker (tamsulosin [TAM], 0.1 and 0.3 mg/kg), a cholinesterase inhibitor (distigmine [DIS], 0.3 and 1.0 mg/kg), or an EP2/3 dual agonist (ONO-8055, 0.01 and 0.03 mg/kg). We compared cystometric parameters after administration of the vehicle and drugs using a paired t test. P < .05 was considered to be statistically significant. RESULTS: Compared with the vehicle, TAM significantly decreased maximum intravesical pressure during voiding (Pmax), while DIS significantly increased it. However, neither drug significantly affected PVR or the residual urine rate (RUR). On the other hand, ONO-8055 significantly decreased PVR and tended to decrease RUR, although it did not significantly affect Pmax. CONCLUSION: The present study was unable to demonstrate that stimulation of EP2 and EP3 receptors caused major improvements in UAB/DU due to DC. However, this equivocal result could arise from inherent limitations of the STZ-induced diabetic rat as a UAB/DU model.

Further characterization of a novel EP2 and EP3 receptor dual agonist, ONO-8055, on lower urinary tract function in normal and lumbar canal stenosis rats.

AIMS: To further explore the effects of a novel EP2 and EP3 dual agonist, ONO-8055, on detrusor contractility, we investigated the responses of bladder strips from sham and lumbar canal stenosis (LCS) rats to this agonist, its effects on lower urinary tract function in normal rats, and mRNA expression of EP2 and EP3 receptors in the sham and LCS rats. METHODS: The responses of bladder strips from sham and LCS rats to ONO-8055 were measured. The effects of ONO-8055 on LUT function of normal rats were investigated with awake cystometry and intraurethral perfusion pressure (Pura) measurements. The relative mRNA of bladder and urethral tissue of the sham and LCS rats was quantified using specific probes for EP1, EP2, EP3, and EP4 genes. RESULTS: Compared with the vehicle, the muscle tensions of both the sham and LCS rats were significantly increased after adding this agonist. On awake cystometry of normal rats, bladder capacity and Pura were decreased in the ONO-8055 groups, but a statistically significant difference in mean changes was demonstrated only between the vehicle group and the group receiving the highest dose. Compared with the sham rats, mRNA expressions of the four EP receptors in the lower urinary tract of the LCS rats did not show a statistically significant difference. CONCLUSIONS: This agonist did not augment bladder contractility or urethral relaxation in normal rats.

Effects of an EP2 and EP3 Receptor Dual Agonist, ONO-8055, on a Radical Hysterectomy-Induced Underactive Bladder Model in Monkeys.

OBJECTIVES: The objective was to develop an underactive bladder (UAB) model in primates and to evaluate the potential of prostanoid EP2 and EP3 receptor dual agonist ONO-8055 to become a therapeutic agent for UAB. METHODS: A surgical procedure resembling radical hysterectomy was performed on female cynomolgus monkeys. Subsequently, in vitro muscle strip studies were performed using bladder muscle strips from normal monkeys and monkeys that underwent surgery. In addition, uroflowmetric data were obtained at specified days after the surgery. To evaluate the effects of ONO-8055 and distigmine (DIS) on voiding function in the UAB monkey model, uroflowmetry was performed approximately 1 week after the surgery, before and after the cumulative intravenous administration of the compounds at 2 h intervals. RESULTS: In the bladder muscle strip studies, the responses to potassium chloride at 2 months, and carbachol and electrical field stimulation from 2 weeks decreased significantly. Voided volume (VV), maximum flow rate (Qmax), and average flow rate (Qave) decreased after surgery, while voiding time (VT) increased. In this model, ONO-8055 and DIS significantly increased VV and Qmax. DIS prolonged VT, while ONO-8055 had no effect. The results also showed that ONO-8055 increased Qave. CONCLUSIONS: We developed a neurogenic UAB model in primates. As ONO-8055 improved voiding function in this model to at least the same degree as DIS, this EP2 and EP3 receptor dual agonist has the potential to be a candidate for neurogenic UAB pharmacotherapy.

Synthesis and evaluation of a potent, well-balanced EP2/EP3 dual agonist.

A highly potent and well-balanced dual agonist for the EP2 and EP3 receptors is described. Optimization of the lead compound was accomplished in consideration of the relative agonist activity against each EP subtype receptor and the pharmacokinetic profile. As the result, 2-[(2-{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-hydroxy-4-methyl-1-penten-1-yl]-5-oxocyclopentyl}eth-yl)thio]-1,3-thiazole-4-carboxylic acid (10) showed excellent potency (human EC50 EP2 = 1.1 nM, EP3 = 1.0 nM) with acceptable selectivity over the EP1 and EP4 subtypes (>2000-fold). Further fine-tuning of compound 10 led to identification of ONO-8055 as a clinical candidate. ONO-8055 was effective at an extremely low dose (0.01 mg/kg, po, bid) in rats, and dose-dependently improved voiding dysfunction in a monkey model of underactive bladder (UAB). ONO-8055 is expected to be a novel and highly promising drug for UAB.

Inhibitory effects of retigabine, a Kv7 channel activator, on mechanosensitive primary bladder afferent activities and nociceptive behaviors in rats.

AIMS: Kv7 voltage-gated potassium channels have been suggested to modulate mechano-afferent transduction and nociception in the bladder. We investigated the effects of retigabine, a Kv7 channel activator, on rhythmic bladder contractions (RBCs), and single-unit afferent activities (SAAs) of the primary bladder mechanosensitive afferent nerve fibers in urethane-anesthetized rats. In addition, the effects of pretreatment with retigabine on the nociceptive behaviors provoked by an intravesical instillation of resiniferatoxin (RTX) were evaluated in the conscious condition. METHODS: Female Sprague-Dawley rats were used. Under urethane anesthesia, saline was instilled into the bladder until RBCs were induced reproducibly. Then, the effects of intravenous, cumulative administrations of retigabine (0.1-3 mg/kg) or vehicle (saline) on RBCs were assessed. In separate animals, SAAs of Aδ- and C-fibers were identified by electrical stimulation of the pelvic nerve and by bladder distention with saline. After baseline recording, vehicle or retigabine (0.01-1 mg/kg) was administered intravenously and further recordings were performed. Under pretreatment with vehicle or retigabine (3 mg/kg intraperitoneally), the frequencies of lower abdominal licking and freezing were counted and scored as the bladder nociceptive behaviors induced by intravesical RTX instillation (3 µM, 0.3 ml). RESULTS: Retigabine dose-dependently decreased both the frequency and the amplitude of RBCs and SAAs of both Aδ- and C-fibers. The effect on RBCs was more potent on the frequency than the amplitude. Retigabine inhibited the RTX-induced abdominal licking, but not freezing. CONCLUSION: Kv7 channels are likely to be implicated in inhibition of bladder mechano- and nociceptive sensory transduction. Neurourol. Urodynam. 36:280-285, 2017. © 2015 Wiley Periodicals, Inc.

Promising Effects of a Novel EP2 and EP3 Receptor Dual Agonist, ONO-8055, on Neurogenic Underactive Bladder in a Rat Lumbar Canal Stenosis Model.

PURPOSE: We investigated whether the novel EP (prostaglandin E2) receptor agonist ONO-8055 would improve the lower urinary tract dysfunction of neurogenic underactive bladder in a rat lumbar spinal canal stenosis model. MATERIALS AND METHODS: First, we studied the agonistic effect of ONO-8055 on EP receptors in EP receptor expressing CHO (Chinese hamster ovary) cells using the increase in the intracellular calcium level and intracellular cAMP (cyclic adenosine monophosphate) production as indicators of receptor activation. The effects of ONO-8055 on bladder and urethral strips from normal rats were then investigated. Finally, the effects of ONO-8055 on bladder and urethral function in rats with lumbar spinal canal stenosis were evaluated by awake cystometry and intraurethral perfusion pressure, respectively. The effects of tamsulosin and distigmine on urethral pressure were also evaluated. RESULTS: ONO-8055 is a highly potent and selective agonist for EP2 and EP3 receptors on CHO cells. While this compound contracted bladder strips, it relaxed urethral strips. Awake cystometry showed that ONO-8055 significantly decreased bladder capacity, post-void residual urine and voiding pressure. Compared with vehicle, tamsulosin and ONO-8055 significantly decreased urethral pressure. CONCLUSIONS: ONO-8055 decreased post-void residual urine, probably by decreasing bladder capacity. The decrease in voiding pressure probably resulted from the lowered urethral pressure due to relaxation of the urethra. Thus, the novel EP2 and EP3 receptor dual agonist ONO-8055 has the potential to improve neurogenic underactive bladder.

Discovery of highly potent dual EP(2) and EP(3) agonists with subtype selectivity.

The cyclic carbamate derivatives, 2-{[2-((4S)-4-{(1E,3R)-8-fluoro-3-hydroxy-4,4-dimethyl-1-octenyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}-1,3-thiazole-4-carboxylic acid (5) and 2-{[2-((4S)-4-{(1E,3R)-3-[1-(4-fluorobutyl)cyclobutyl]-3-hydroxy-1-propenyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}-1,3-thiazole-4-carboxylic acid (7) were identified as the first potent dual EP2 and EP3 agonists with selectivity against the EP1 and EP4 subtypes. Compounds 5 and 7 demonstrated highly potent dual EP2 and EP3 agonist activity with EC50 values of 10nM or less. In addition, these compounds possess structural features distinct from natural prostaglandins, such as a cyclic carbamate moiety, a dimethyl or cyclobutyl group and a terminal fluorine atom.

Effects of α1 antagonist and cholinesterase inhibitor on cystometric parameters in lumbar canal stenosis rats with underactive bladder.

OBJECTIVE: To examine the lower urinary tract function of a rat lumbar canal stenosis (LCS) model by in vivo cystometry before and after α1 adrenoceptor antagonist or cholinesterase inhibitor administration. MATERIALS AND METHODS: One small hole was drilled at the fifth lumbar vertebral arch, and a rectangular piece of silicone rubber was inserted into the L5-L6 epidural space. Two weeks after the surgery, awake cystometry was performed before and after the oral administration of the vehicle, tamsulosin (TAM, 0.03 and 0.1 mg/kg), or distigmine (DIS, 0.3 and 1 mg/kg). We compared the awake cystometry parameters before and after drug administration. RESULTS: The LCS rats showed a large maximum cystometric capacity (MCC) and a high residual urine rate with a lower maximum bladder pressure during micturition (Pmax). TAM and DIS significantly decreased the pressure at the onset of voiding contraction, MCC, and postvoid residual urine volume. Residual urine rate was also significantly decreased by DIS at 0.3 and 1.0 mg/kg, and TAM at 0.03 mg/kg. DIS significantly increased the frequency of nonvoiding contractions per minute. Pmax was not significantly different even after administration of DIS. CONCLUSION: The LCS rats had salient characteristics of severe infra-sacral neuropathic bladder dysfunction. TAM and DIS decreased postvoid residual urine volume, but this decrease was not accompanied by an increased Pmax or increased voided volume. Rather, decreased MCC was a possible contributing factor. Moreover, increased nonvoiding contractions after administration of DIS might participate in the decreased MCC. This novel model will be useful in studying the pharmacotherapy of the underactive bladder.

A novel animal model of underactive bladder: analysis of lower urinary tract function in a rat lumbar canal stenosis model.

AIMS: An animal model of neurogenic underactive bladder (UAB) has not been established. It was reported that a rat lumbar spinal canal stenosis (LCS) model created by cauda equina compression manifested intermittent claudication and allodynia. In this study, we examined the lower urinary tract function of the rat LCS model. METHODS: One small hole was drilled at the fifth lumbar vertebral arch (sham), and a rectangular piece of silicone rubber was inserted into the L5-L6 epidural space (LCS). Before and after surgery, a metabolic cage study was performed. After surgery, awake cystometry (CMG) and an in vitro muscle strip study were performed. Bladder morphology was evaluated by hematoxylin and eosin staining. RESULTS: The LCS rats showed a significant decrease in voided volume and a significant increase in postvoid residual volume and residual urine rate compared with Sham rats. CMG showed that the postvoid residual urine volume and numbers of non-voiding contractions significantly increased, while the voided volume, threshold pressure, and maximum intravesical pressure during voiding significantly decreased. There were no significant differences between sham and LCS rats in response to carbachol. In contrast, there was a significant increase in response to field stimulation, especially at lower frequencies, in LCS rats. LCS rats showed no obvious difference in detrusor morphology. CONCLUSIONS: This rat model requires a relatively simple surgical procedure and has characteristics of neurogenic UAB. It seems to be useful in the pathophysiological elucidation of UAB and might have potential for assessment of pharmacotherapy of UAB.

ONO-8130, a selective prostanoid EP1 receptor antagonist, relieves bladder pain in mice with cyclophosphamide-induced cystitis.

Given the previous evidence for involvement of prostanoid EP1 receptors in facilitation of the bladder afferent nerve activity and micturition reflex, the present study investigated the effect of ONO-8130, a selective EP1 receptor antagonist, on cystitis-related bladder pain in mice. Cystitis in mice was produced by intraperitoneal administration of cyclophosphamide at 300mg/kg. Bladder pain-like nociceptive behavior and referred hyperalgesia were assessed in conscious mice. Phosphorylation of extracellular signal-regulated kinase (ERK) in the L6 spinal cord was determined by immunohistochemistry in anesthetized mice. Cyclophosphamide treatment caused bladder pain-like nociceptive behavior and referred hyperalgesia accompanying cystitis symptoms, including increased bladder weight and vascular permeability and upregulation of cyclooxygenase-2 in the bladder tissue. Oral preadministration of ONO-8130 at 0.3-30 mg/kg strongly prevented both the bladder pain-like behavior and referred hyperalgesia in a dose-dependent manner, but had slight effect on the increased bladder weight and vascular permeability. Oral ONO-8130 at 30 mg/kg also reversed the established cystitis-related bladder pain. Intravesical administration of prostaglandin E2 caused prompt phosphorylation of ERK in the L6 spinal cord, an effect blocked by ONO-8130. Our findings strongly suggest that the prostaglandin E2/EP1 system participates in processing of cystitis-related bladder pain, and that EP1 antagonists including ONO-8130 are useful for treatment of bladder pain, particularly in interstitial cystitis. Prostaglandin E2 contributes to cystitis-related bladder pain via EP1 receptors in mice, indicating possible therapeutic usefulness of selective EP1 antagonists.

The potent inducible nitric oxide synthase inhibitor ONO-1714 inhibits neuronal NOS and exerts antinociception in rats.

We evaluated if ONO-1714, known as an inducible nitric oxide synthase (iNOS) inhibitor, could inhibit neuronal NOS (nNOS) and exert antinociception. ONO-1714 potently inhibited both crude rat cerebellar NOS and recombinant human nNOS in vitro. Systemic ONO-1714 at 1-10 mg/kg suppressed carrageenan-induced thermal hyperalgesia in rats, an effect being equivalent to the antinociception caused by L-NAME or 7-nitroindazole at 25 mg/kg. The same doses of ONO-1714 also caused hypertension. Intrathecal (i.t.) ONO-1714 potently reduced the hyperalgesia, the effective dose range (0.2-0.6 microg/rat) being much lower than the antinociceptive dose (150 microg/rat) of i.t. L-NAME. Thus, ONO-1714 is considered a potent inhibitor of nNOS in addition to iNOS. The distinct relative antinociceptive activities of systemic and i.t. ONO-1714 are attributable to its possible poor blood-brain barrier permeability.

Effect of a potent iNOS inhibitor (ONO-1714) on acetaminophen-induced hepatotoxicity in the rat.

Overproduction of nitric oxide (NO) in the liver has been implicated as an important event in endotoxin shock and in other models of hepatic inflammation and injury. The present study was undertaken to evaluate the effect of ONO-1714, a potent and specific inhibitor of inducible NO synthase (iNOS), on acetaminophen-induced hepatotoxicity in the rats. Oral administration of ONO-1714 dose-dependently inhibited NOx (NO2- and NO3-) accumulation in rat plasma after lipopolysaccharide (LPS) treatment. Intraperitoneal acetaminophen at 1 g/kg caused damage to the centrilobular regions of the liver and increase in serum alanine and aspartate transaminase (ALT and AST, respectively) levels accompanied by elevated plasma NOx levels after 24 h. Oral administration of ONO-1714 at 10 and 100 microg/kg dose-dependently reduced the acetaminophen-induced hepatic tissue damage and the increases in serum ALT and AST levels. ONO-1714 also blocked the increase in plasma NOx concentrations. These findings demonstrate that oral ONO-1714, an iNOS inhibitor, protects against acetaminophen-evoked hepatic inflammation/injury, strongly suggesting that NO produced by iNOS plays a key role in the pathogenesis of this drug-induced hepatotoxicity.